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A Library Construction Method for Single-Cell Whole-Genome Bibisulfite Sequencing

A bisulfite and construction method technology, applied in the field of library construction of single-cell whole-genome bisulfite sequencing, can solve the problems of low data comparison rate, etc. The effect of improving the data comparison rate

Active Publication Date: 2021-08-13
ZHEJIANG ANNOROAD BIO TECH CO LTD +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0013] However, in the experimental analysis results made by this method, the data comparison rate (Unique Mapped Ratio) is low

Method used

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  • A Library Construction Method for Single-Cell Whole-Genome Bibisulfite Sequencing
  • A Library Construction Method for Single-Cell Whole-Genome Bibisulfite Sequencing
  • A Library Construction Method for Single-Cell Whole-Genome Bibisulfite Sequencing

Examples

Experimental program
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Embodiment 1

[0069] The present invention will be described more specifically by way of examples below. It should be understood that the embodiments described here are used to explain the present invention, not to limit the present invention.

[0070] 1. Collection of Single Cells

[0071] Single cells were collected in a volume within 0.5 μL into 0.2 mL thin-walled PCR tubes containing 1 μL of calcium and magnesium-free PBS buffer. The collected single cells are named according to the following format.

[0072]

[0073] 2. Cell Lysis

[0074] Prepare Lysis Buffer 10mM Tris-Cl (pH 7.4), 2% SDS. The single-cell sample in a 0.2mL centrifuge tube has a volume of about 1μL. No need to transfer the tube, directly add 11.5μL Lysis Buffer and 0.5μL proteinase K to the tube, vortex slightly to mix, and then centrifuge. React for 1 hour at 37°C on a PCR instrument, and add ddH after the reaction 2 O to a total volume of 20 μL.

[0075] 3. Bisulfite treatment

[0076] The sample obtained i...

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Abstract

The invention provides a method for constructing a single-cell whole-genome bisulfite sequencing library. Specifically, it relates to an improved method for constructing a library for bisulfite sequencing. The library construction method includes: single cell lysis; bisulfite treatment; one-strand and two-strand synthesis using the bisulfite treatment product as a template; and constructing a library for sequencing using the second-strand synthesis product as a template. Wherein, in the purification step after bisulfite treatment, tRNA is added to improve the purification and recovery efficiency of the treated product. By adopting the library construction method of the present invention, the loss of samples is reduced, and the comparison rate of sequencing data is improved.

Description

technical field [0001] The invention belongs to the field of gene detection, and in particular relates to a library construction method for single-cell whole-genome bisulfite sequencing. Background technique [0002] DNA methylation is an important epigenetic modification of eukaryotic genomic DNA, that is, under the action of DNA methyltransferase (DNA methyltransferase, DNMT), S-adenosylmethylthio The process in which the methyl group of amino acid (SAM) is covalently bonded to cytosine of DNA molecule to form 5-methylcytosine (5-mC). DNA methylation plays an important role in the maintenance of normal cell function in higher organisms, genetic imprinting, embryonic development, aging and the occurrence of human tumors and other biological processes. In invertebrates, genomic DNA methylation participates in regulating the adaptation process of the organism to the environment by regulating the expression pattern of genes. Therefore, obtaining the methylation data of all c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C40B50/06C12N15/10
CPCC12Q1/6806C12Q1/6869C40B50/06C12Q2531/113C12Q2535/122C12Q2523/125C12Q2563/143C12Q2563/149
Inventor 柳青王晓雯洪燕赵红梅玄兆伶李大为梁峻彬陈重建
Owner ZHEJIANG ANNOROAD BIO TECH CO LTD
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